This study shows that the main epithelial cells of the renal tubules (Malpighian tubules), the principal cells, express both DILP5 and the DTK receptor DTKR, suggesting that these insulin-producing cells are targets of circulating DTKs. Indeed, it was found that DTK signaling regulates levels of DILP5 in principal cells under nutritional stress. Since the renal tubules are not innervated, DTK can only reach them as a circulating hormone, likely to be released from the intestine (Söderberg, 2011).

In Drosophila the renal tubules display high metabolic activity and play roles, not only in water and ion transport, but also in oxidative stress, detoxification and immune responses. Encouraged by this and by the likely importance of insulin signaling in the physiology of the kidneys of mammals, the roles were investigated of DILP5 signaling locally in the renal tubules. Interference with the expression levels of DTKR, DILP5, dInR and some further components of the insulin-signaling pathway in principal cells during metabolic and oxidative stress all lead to altered lifespan. Furthermore, knockdown of superoxide dismutase (SOD2) in principal cells leads to decreased lifespan at desiccation and oxidative stress, suggesting a possible link between insulin signaling and oxidative stress responses. It is proposed that insulin signaling in the tubules may be part of an autocrine regulation of renal function that in turn is controlled by hormonal DTK signaling from the intestine at metabolic and oxidative stress (Söderberg, 2011).

This study has identified the renal tubules as a novel site of insulin production and signaling in Drosophila. The principal cells of these tubules produce DILP5 and express the ubiquitous DILP receptor, dInR. From these findings it is suggested that DILP5 may signal locally within the epithelium of the renal tubules. This local DILP signaling appears to be under hormonal regulation during desiccative, nutritional and oxidative stress by means of the peptide DTK acting on the receptor, DTKR, localized on the principal cells. These findings, that diminished DTKR, DILP5 and dInR extend life span, suggest an involvement of this signaling pathway in tubules in desiccation, nutritional and oxidative stress responses in adult Drosophila. Finally, manipulations of dS6K, 4E-BP and SOD (SOD2) in principal cells altered life span of flies at stress supporting that insulin signaling acts within the tubules, probably in regulation of oxidative stress responses. Interestingly, the signaling within the renal tubules affects the survival of the whole organism as shown also for mitochondrial function in tubules at oxidative stress (Söderberg, 2011).

The roles of DILPs in stress resistance and regulation of life span are well established in Drosophila, but hormonal mechanisms for regulation of production and release of DILPs in IPCs of adult flies have not been reported. Thus this demonstration of DTKs acting on IPCs in the renal tubules is a first identification of a hormonal factor regulating DILP release in adult insects. Interestingly, there is evidence for actions of tachykinins on IPCs also in mammals: the tachykinin substance P has been shown to increase insulin secretion from the pancreas of rat and pig and this effect is reversed in the diabetic rat (Schmidt, 2000; Adeghate, 2001; Söderberg, 2011 and references therein).

Since the renal tubules are not innervated, peptide receptors in this tissue can only be activated by hormonal messengers. One source of hormonal DTKs in Drosophila is a population of endocrine cells in the intestine (midgut) located close to the attachment of the renal tubules (Siviter, 2000). In locust and cockroach similar cells have been identified and it was shown that at starvation tachykinin-related peptide was released into the circulation (Söderberg, 2011).

Renal tubules in insects have been primarily investigated with respect to their function in water and ion transport and several peptide hormones have been implicated in the control of diuresis. The current findings suggest that peptide hormones that target the renal tubules may play roles other than in direct regulation of diuresis. The Drosophila renal tubules express an impressive array of genes and combined with experimental analysis it is suggestive that this tissue partakes in detoxification processes, oxidative stress, dietary osmotic stress and immune responses (Söderberg, 2011 and references therein).

How does DTK signaling to the renal tubules produce a response that affects sensitivity to desiccation and starvation? The DTK signal may be a general metabolic stress signal that reaches the renal tubules. In these experiments this stress signaling is amplified with the over-expression of DTKR in principal cells and diminished by its knockdown leading to changes in lifespan. The role of DTK may be to regulate factors in principal cells involved in local metabolism, oxidative stress resistance or immune responses at the cost of decreased life span when in over-drive. One such a factor may be DILP5. Both in Drosophila and C. elegans immune response genes are expressed in the intestine (including renal tubules in the fly) and recent work has shown that these genes are under control of insulin signaling. In Drosophila the DILP signaling pathway is involved in infection-induced wasting (loss of energy stores) where reduced signaling leads to reduction in pathology (Söderberg, 2011).

Also oxidative stress resistance is linked to insulin signaling in Drosophila. Superoxide dismutases (SOD) are key enzymes protecting proteins from reactive oxygen species and are thought to be regulated by insulin signaling: SOD activity is elevated in chico (dInR substrate) mutants of Drosophila and Daf-2 mutants of C. elegans. Also in yeast insulin-signaling mutations affect lifespan via SOD. The knockdown of Sod2 (encoding MnSOD), but not Sod1, in renal tubules decreased lifespan at desiccation and oxidative stress in Drosophila. Thus, it is possible that DILP signaling in tubules target mitochondrial SOD2 and affects resistance to oxidative stress. Interestingly, diminishing oxidative stress resistance via Sod2 locally in the principal cells of Drosophila renal tubules is sufficient to shorten the lifespan of the fly during stress. This is similar to findings in a study of genetical impairment of a mitochondrial inner membrane ATP/ADP exchanger in the same cells (Söderberg, 2011),

In conclusion, this study presents evidence for DTK controlled insulin signaling in the renal tubules of Drosophila being important for survival at metabolic and oxidative stress. The findings of this study may suggest an autocrine regulatory loop within the tubules with a role in renal function. Local signaling within Drosophila renal tubules has previously been demonstrated with endogenously produced tyramine and nitric oxide, that regulate chloride permeability and innate immune responses, respectively. It is possible that the insulin signaling in the renal tubules is part of the epithelial immune system or oxidative stress defense via SOD, but it cannot be excluded that the dInRs on principal cells regulate DILP5 production or release and that additional DILP5 targets are located outside the renal tubules (Söderberg, 2011).

Control of lipid metabolism by Tachykinin in Drosophila

The intestine is a key organ for lipid uptake and distribution, and abnormal intestinal lipid metabolism is associated with obesity and hyperlipidemia. Although multiple regulatory gut hormones secreted from enteroendocrine cells (EEs) regulate systemic lipid homeostasis, such as appetite control and energy balance in adipose tissue, their respective roles regarding lipid metabolism in the intestine are not well understood. This study demonstrates that Tachykinins (TKs), one of the most abundant secreted peptides expressed in midgut EEs, regulate intestinal lipid production and subsequently control systemic lipid homeostasis in Drosophila and that TKs repress lipogenesis in enterocytes (ECs) associated with TKR99D receptor and protein kinase A (PKA) signaling. Interestingly, nutrient deprivation enhances the production of TKs in the midgut. Finally, unlike the physiological roles of TKs produced from the brain, gut-derived TKs do not affect behavior, thus demonstrating that gut TK hormones specifically regulate intestinal lipid metabolism without affecting neuronal functions (Song, 2014).

Previous studies in mammals have indicated that a few gut secretory hormones, like GLP1 and GLP2, are involved in intestinal lipid metabolism. However, due to gene and functional redundancy, mammalian genetic models for gut hormones and/or their receptors with severe metabolic defects are not available. This study has establish that Drosophila TKs produced from EEs coordinate midgut lipid metabolic processes. The studies clarify the roles of TK hormones in intestinal lipogenesis and establish Drosophila as a genetic model to study the regulation of lipid metabolism by gut hormones (Song, 2014).

Six mature TKs, TK1-TK6, are processed and secreted from TK EEs in both the brain and midgut (Reiher, 2011). Using a specific Gal4 driver line, gene expression in TK EEs was specifically manipulated, leading to the demonstration that loss of gut TKs results in an increase in midgut lipid production. Further, this study showed that TKs regulate intestinal lipid metabolism associated with TKR99D, but not TKR86C, which is consistent with the expression of these receptors. Consistent with previous reports that TK/TKR99D signaling regulates cAMP level and PKA activation, loss of gut TKs is associated with a reduction in PKA activity in ECs, and overexpression of a PKA catalytic subunit was able to reverse the increased intestinal lipid production associated with loss of TKR99D. In addition, the transcription factor SREBP that triggers lipogenesis was controlled by TK/TKR99D/PKA signaling. Taken together, these results suggest that TKs produced from EEs regulate midgut lipid metabolism via TKR99D/PKA signaling and regulation of, at least, SREBP-induced lipogenesis in ECs (Song, 2014).

Interestingly, this study reveals that TKs derived from either the brain or gut exhibit distinct functions: TKs derived from gut control intestinal lipid metabolism, whereas TKs derived from brain control behavior. This is reminiscent of the distinct functions of mammalian secreted regulatory peptides, where different spatial expressions or deliveries of peptides like Ghrelin can result in distinct physiological functions. In addition, some prohormones encode multiple mature peptides that can have multiple functions. For example, processing of proglucagon in the pancreas α cells preferentially gives rise to glucagon, which antagonizes the effect of insulin. In intestine L cells, however, proglucagon is mostly processed into GLP1 to promote insulin release. These studies of TKs exemplify how secreted regulatory peptides derived from different tissues can be associated with fundamentally diverse physiological functions. Clearly, additional studies examining the function of secreted peptides in a cell-type- and tissue-specific manner are needed to fully appreciate and unravel their complex roles both in flies and mammals (Song, 2014).

There is a growing body of studies emphasizing that intestinal lipid metabolism is key to the control of systemic lipid homeostasis. For example, chemicals such as orlistat, designed to inhibit dietary lipid digestion/absorption in the intestine, efficiently reduce obesity. In addition, mammalian inositol-requiring enzyme 1β deficiency-induced abnormal chylomicron assembly in the small intestine results in hyperlipidemia. Similarly, in Drosophila, dysfunction of intestinal lipid digestion/absorption caused by Magro/LipA deficiency eventually decreases whole-body lipid storage and starvation resistance in Drosophila. Further, intestinal lipid transport, controlled by lipoproteins, is essential for systemic lipid distribution and energy supply in other tissues. Consistent with these observations, this study demonstrates that increased midgut lipid synthesis associated with gut TK deficiency is sufficient to elevate systemic lipid storage. Although TK ligands and TK receptors show high homologies between mammals and fruit flies, whether mammalian TK signaling plays a similar role in intestinal lipid metabolism is largely unknown. Future studies will reveal whether mammalian TK signaling affects intestinal lipid metabolism as in Drosophila. If this is the case, it may provide a therapeutic opportunity for the treatment of intestinal lipid metabolic disorder and obesity (Song, 2014).

Production and secretion of gut hormones are precisely regulated under various physiological conditions. Similar to previous observations that starvation induces gut TK secretion in other insects, this study found that nutrient deprivation promotes TK production in EEs. Interestingly, feeding of amino-acid-enriched yeast, but not coconut oil or sucrose, potently suppressed gut TK levels, indicating that amino acids may act directly on TK production in EEs. It has been reported that dietary nutrients regulate gut hormone production through certain receptors located on the cell membrane of EEs in mammals. Future studies will be necessary to elucidate the detailed mechanism by which nutrients regulate TK production from EEs (Song, 2014).

Pain signaling in vertebrates is modulated by neuropeptides like Substance P (SP). To determine whether such modulation is conserved and potentially uncover novel interactions between nociceptive signaling pathways SP/Tachykinin signaling was examined in a Drosophila model of tissue damage-induced nociceptive hypersensitivity. Tissue-specific knockdowns and genetic mutant analyses revealed that both Tachykinin and Tachykinin-like receptor (DTKR99D) are required for damage-induced thermal nociceptive sensitization. Electrophysiological recording showed that DTKR99D is required in nociceptive sensory neurons for temperature-dependent increases in firing frequency upon tissue damage. DTKR overexpression caused both behavioral and electrophysiological thermal nociceptive hypersensitivity. Hedgehog, another key regulator of nociceptive sensitization, was produced by nociceptive sensory neurons following tissue damage. Surprisingly, genetic epistasis analysis revealed that DTKR function was upstream of Hedgehog-dependent sensitization in nociceptive sensory neurons. These results highlight a conserved role for Tachykinin signaling in regulating nociception and the power of Drosophila for genetic dissection of nociception (Im, 2015).

This study establishes that Tachykinin signaling regulates UV-induced thermal allodynia in Drosophila larvae. It is envisioned that UV radiation either directly or indirectly activates Tachykinin expression and/or release from peptidergic neuronal projections - likely those within the CNS that express DTK and are located near class IV axonal tracts. Following release, it is speculated that Tachykinins diffuse to and ultimately bind DTKR on the plasma membrane of class IV neurons. This activates downstream signaling, which is mediated at least in part by a presumed heterotrimer of α G alpha (Gαq, CG17760), a G β (Gβ5), and a G γ (Gγ1) subunit. One likely downstream consequence of Tachykinin receptor activation is Dispatched-dependent autocrine release of Hh from these neurons. It is envisioned that Hh then binds to Patched within the same class IV neurons, leading to derepression of Smo and activation of downstream signaling through this pathway. One new aspect of the thermal allodynia response dissected in this study is that the transcription factors Cubitius interruptus and Engrailed act downstream of Smo, suggesting that, as in other Hh-responsive cells, activation of target genes is an essential component of thermal allodynia. Finally, activation of Smo impinges upon Painless through as yet undefined mechanisms to regulate thermal allodynia. Some of the implications of this model for Tachykinin signaling, Hh signaling, and their conserved regulation of nociceptive sensitization are discussed below (Im, 2015).

The results demonstrate that Tachykinin is required for UV-induced thermal allodynia. UV radiation may directly or indirectly trigger Tachykinin expression and/or release from the DTK-expressing neurons. Given the transparent epidermis and cuticle, direct induction mechanisms are certainly plausible. Indeed in mammals, UV radiation causes secretion of SP and CGRP from both unmyelinated c fibers and myelinated Aγ fibers nociceptive sensory afferents. Furthermore, in the Drosophila intestine Tachykinin release is induced by nutritional and oxidative stress, although the effect of UV has not been examined. The exact mechanism of UV-triggered neuropeptide release remains unclear; however, it is speculated that UV causes depolarization and activation of exocytosis of Tachykinin-containing vesicles (Im, 2015).

In heterologous cells synthetic Tachykinins (DTK1-5) can activate DTKR. Immunostaining analysis of dTk and genetic analysis of tissue-specific function of dtkr supports the model that Tachykinins from brain peptidergic neurons bind to DTKR expressed on class IV neurons. Pan-neuronal, but not class IV neuron- specific knockdown of dTk reduced allodynia, whereas modulation of DTKR function in class IV neurons could either decrease (RNAi) or enhance (overexpression) thermal allodynia. How do brain-derived Tachykinins reach DTKR expressed on the class IV neurons? The cell bodies and dendritic arbors of class IV neurons are located along the larval body wall, beneath the barrier epidermal cells. However, the axonal projection of each nociceptive neuron extends into the ventral nerve cord (VNC) of the CNS in close proximity to Tachykinin-expressing axons. Because neuropeptide transmission does not depend on specialized synaptic structures, it is speculated given their proximity that Tachykinin signaling could occur via perisynaptic or volume transmission. An alternative possibility is that Tachykinins are systemically released into the circulating hemolymph as neurohormones following UV irradiation, either from the neuronal projections near class IV axonal tracts or from others further afield within the brain (Im, 2015).

Indeed the gain-of-function behavioral response induced by overexpression of DTKR, a receptor that has not been reported to have ligand-independent activity, suggests that class IV neurons may be constitutively exposed to a low level of subthreshold DTK peptide in the absence of injury. The direct and indirect mechanisms of DTK release are not mutually exclusive and it will be interesting to determine the relative contribution of either mechanism to sensitization (Im, 2015).

Like most GPCRs, DTKR engages heterotrimeric G proteins to initiate downstream signaling. Gq/11 and calcium signaling are both required for acute nociception and nociceptive sensitization. The survey of G protein subunits identified a putative Gαq, CG17760. It was demonstrated that DTKR activation leads to an increase in Ca2+, strongly pointing to Gαq as a downstream signaling component. To date, CG17760 is one of three G alpha subunits encoded in the fly genome that has no annotated function in any biological process. For the G β and G γ classes, Gβ5 and Gγ1 were identified. Gβ5 was one of two G β subunits with no annotated physiological function. Gγ1 regulates asymmetric cell division and gastrulation, cell division, wound repair, and cell spreading dynamics. The combination of tissue-specific RNAi screening and specific biologic assays, as employed in this study, has allowed assignment of a function to this previously 'orphan' gene in thermal nociceptive sensitization. The findings raise a number of interesting questions about Tachykinin and GPCR signaling in general in Drosophila: Are these particular G protein subunits downstream of other neuropeptide receptors? Are they downstream of DTKR in biological contexts other than pain? Could RNAi screening be used this efficiently in other tissues/behaviors to identify the G protein trimers relevant to those processes (Im, 2015)?

To date three signaling pathways were found that regulate UV-induced thermal allodynia in Drosophila: TNF, Hedgehog, and Tachykinin. All are required for a full thermal allodynia response to UV but genetic epistasis tests reveal that TNF and Tachykinin act in parallel or independently, as do TNF and Hh. This could suggest that in the genetic epistasis contexts, which rely on class IV neuron-specific pathway activation in the absence of tissue damage, hyperactivation of one pathway (say TNF or Tachykinin) compensates for the lack of the function normally provided by the other parallel pathway following tissue damage. While TNF is independent of Hh and DTKR, analysis of DTKR versus Hh uncovered an unexpected interdependence (Im, 2015).

This study showed that Hh signaling is downstream of DTKR in the context of thermal allodynia. Two pieces of genetic evidence support this conclusion. First, flies transheterozygous for dTk and smo displayed attenuated UV-induced thermal allodynia. Thus, the pathways interact genetically. Second, and more important for ordering the pathways, loss of canonical downstream Hh signaling components blocked the ectopic sensitization induced by DTKR overexpression. It was previously shown that loss of these same components also blocks allodynia induced by either UV or Hh hyperactivation (Babcock, 2011), suggesting that these downstream Hh components are also downstream of DTKR. The fact that Smo is activated upon overexpression of DTKR within the same cell argues that class IV neurons may need to synthesize their own Hh following a nociceptive stimulus such as UV radiation. The data supporting an autocrine model of Hh production are three fold: (1) only class IV neuron-mediated overexpression of Hh caused thermal allodynia suggesting this tissue is fully capable of producing active Hh ligand; (2) expression of UAS-dispRNAi within class IV neurons blocked UV- and DTKR-induced thermal allodynia, implicating a role for Disp-driven Hh secretion in these cells, and (3) the combination of UAS-dispRNAi and UV irradiation caused accumulation of Hh punctae within class IV neurons. Disp is not canonically viewed as a downstream target of Smo and indeed, blocking disp did not attenuate UAS-PtcDN-induced or UAS-TNF-induced allodynia, indicating that Disp is specifically required for Hh production between DTKR and Smo. Thus, Tachykinin signaling leads to Hh expression, Disp-mediated Hh release, or both (Im, 2015).

Autocrine release of Hh has only been demonstrated in a few non-neuronal contexts to date. This signaling architecture differs from what has been found in Drosophila development in two main ways. One is that DTKR is not known to play a patterning role upstream of Smo. The second is that Hh-producing cells are generally not thought to be capable of responding to Hh during the formation of developmental compartment boundaries (Im, 2015).

What happens downstream of Smoothened activation to sensitize class IV neurons? Ultimately, a sensitized neuron needs to exhibit firing properties that are different from those seen in the naïve or resting state. Previously, sensitization was examined only at the behavioral level. This study also monitored changes through extracellular electrophysiological recordings. These turned out to correspond remarkably well to behavioral sensitization. In control UV-treated larvae, nearly every temperature in the low 'allodynic' range showed an increase in firing frequency in class IV neurons upon temperature ramping. Dtkr knockdown in class IV neurons abolished the UV-induced increase in firing frequency seen with increasing temperature and overexpression of DTKR increased the firing rate comparable to UV treatment. This latter finding provides a tidy explanation for DTKR-induced 'genetic allodynia.' The correspondence between behavior and electrophysiology argues strongly that Tachykinin directly modifies the firing properties of nociceptive sensory neurons in a manner consistent with behavioral thermal allodynia (Im, 2015).

Genetically, knockdown of painless blocks DTKR- or PtcDN-induced ectopic sensitization suggesting that, ultimately, thermal allodynia is mediated in part via this channel. Indeed, the SP receptor Neurokinin-1 enhances TRPV1 function in primary rat sensory neurons. Tachykinin/Hh activation could lead to increased Painless expression, altered Painless localization, or to post-translational modification of Painless increasing the probability of channel opening at lower temperatures. Because thermal allodynia evoked by UV and Hh-activation requires Ci and En, the possibility is favored that sensitization may involve a simple increase in the expression level of Painless, although the above mechanisms are not mutually exclusive. Altered localization has been observed with a different TRP channel downstream of Hh stimulation; Smo activation leads to PKD2LI recruitment to the primary cilium in fibroblasts, thus regulating local calcium dynamics of this compartment. The exact molecular mechanisms by which nociceptive sensitization occurs is the largest black box in the field and will take a concerted effort by many groups to precisely pin down (Im, 2015).

Tachykinin and Substance P as regulators of nociception: What is conserved and what is not? The results establish that Tachykinin/SP modulation of nociception is conserved across phyla. However, there are substantial differences in the architecture of this signaling axis between flies and mammals. In mammals, activation of TRP channels in the periphery leads to release of SP from the nerve termini of primary afferent C fibers in the dorsal horn. SP and spinal NK-1R have been reported to be required for moderate to intense baseline nociception and inflammatory hyperalgesia although some discrepancies exist between the pharmacological and genetic knockout data. The most profound difference of Drosophila Tachykinin signaling anatomically is that DTK is not expressed and does not function in primary nociceptive sensory neurons. Rather, DTK is expressed in brain neurons and the larval gut, and DTKR functions in class IV neurons to mediate thermal pain sensitization. Indeed, this raises an interesting possibility for mammalian SP studies, because nociceptive sensory neurons themselves express NK-1R and SP could conceivably activate the receptor in an autocrine fashion. A testable hypothesis that emerges from these studies is that NK-1R in vertebrates might play a sensory neuron-autonomous role in regulating nociception. This possibility, while suggested by electrophysiology and expression studies, has not been adequately tested by genetic analyses in mouse to date (Im, 2015).

In summary, this study discovered a conserved role for systemic Tachykinin signaling in the modulation of nociceptive sensitization in Drosophila. The sophisticated genetic tools available in Drosophila have allowed uncovering both a novel genetic interaction between Tachykinin and Hh signaling and an autocrine function of Hh in nociceptive sensitization. This work thus provides a deeper understanding of how neuropeptide signaling fine-tunes an essential behavioral response, aversive withdrawal, in response to tissue damage (Im, 2015).

Drosophila insulin-like peptides (DILPs) play important hormonal roles in the regulation of metabolic carbohydrates and lipids, but also in reproduction, growth, stress resistance and aging. In spite of intense studies of insulin signaling in Drosophila the regulation of DILP production and release in adult fruit flies is poorly understood. This study investigated the role of Drosophila tachykinin-related peptides (DTKs) and their receptors, DTKR (Tachykinin-like receptor at 99D) and NKD (Naked cuticle), in the regulation of brain insulin-producing cells (IPCs) and aspects of DILP signaling. First, DTK-immunoreactive axon terminations were shown close to the presumed dendrites of the IPCs, and DTKR immunolabeling was demonstrated in these cells. Second, targeted RNA interference was used to knock down expression of the DTK receptor, DTKR, in IPCs and the effects the on Dilp transcript levels were monitored in the brains of fed and starved flies. Dilp2 and Dilp3, but not Dilp5, transcripts were significantly affected by DTKR knockdown in IPCs, both in fed and starved flies. Both Dilp2 and Dilp3 transcripts increased in fed flies with DTKR diminished in IPCs whereas at starvation the Dilp3 transcript plummeted and Dilp2 increased. Trehalose and lipid levels were measured as well as survival in transgene flies at starvation. Knockdown of DTKR in IPCs leads to increased lifespan and a faster decrease of trehalose at starvation but has no significant effect on lipid levels. Finally, IPCs were targeted with RNAi or ectopic expression of the other DTK receptor, NKD, but no effect was found on survival at starvation. These results suggest that DTK signaling, via DTKR, regulates the brain IPCs (Birse, 2011).

This study investigated the effects of DTK signaling to IPCs in the Drosophila brain by monitoring Dilp transcript levels and survival at starvation, as well as trehalose and lipid levels in fed and starved flies. The brain IPCs are presumed to release DILP2, DILP3 and DILP5, orthologs of mammalian insulins. Since these insulin-like peptides have been shown to play a significant role in lifespan, in nutritional stress responses and in metabolic regulation, the DTK signaling onto these cells may be of significance for the regulation of vital physiological functions. However, the IPCs are also known to regulate feeding behavior, locomotor activity, sleep-wakefulness and ethanol sensitivity, and they may do so independent of the insulin signaling pathway. Thus, activation or inhibition of signaling in the IPCs may result in actions that are non-insulin mediated, either via other messengers released by the same cells or indirectly by the action of DILPs on specific neurons (see Root, 2011; Birse, 2011 and references therein).

The most direct evidence that DTK signaling affects the brain IPCs is that knock down of the receptor DTKR in IPCs leads to altered expression levels of Dilp2 and Dilp3 transcripts in fed flies and that Dilp3 RNA drops drastically in knockdown flies after 24 h starvation whereas Dilp2 levels increase. Interestingly, the Dilp5 transcript is not affected by DTKR-RNAi, but is the only one that seems affected by starvation both in controls and knockdown flies. It is known that restricted diet conditions in adult (control) flies alter the transcript level of Dilp5, but not Dilp2 and Dilp3 (Broughton, 2010), corroborating the current findings. These data are the first to quantify Dilp transcripts at complete starvation in adults, but an earlier report monitored Dilp transcripts by in situ hybridization of fed and starved third instar larvae. That study noted decreased Dilp3 and Dilp5 transcripts but unaffected levels of Dilp2. This difference could be either dependent on a difference in larval and adult functions of the IPCs or could be due to the difference in techniques used for monitoring transcript levels. Certainly the feeding behavior and metabolism differs greatly between larvae and adults in Drosophila. It should be noted here that insulin expression/signaling also involves autocrine or paracrine feedbacks so that DILP3 may act in stimulatory regulation of expression of DILP2 and DILP5 in the IPCs (Broughton, 2008; Grönke, 2010) whereas DILP6 released from the fat body may negatively regulate the IPCs (Birse, 2011).

Unfortunately there are no reports that unequivocally demonstrate the release of DILPs into the circulation of Drosophila in a quantitative fashion. Thus, indirect measurements, such as Dilp transcript or DILP peptide-immunofluorescence levels in cell bodies of IPCs, have to be matched against the physiological effects seen after manipulations of IPCs. A few indicators of altered insulin signaling have been used here: levels of carbohydrate and lipids as well as effects on lifespan at starvation. As one of the functions of DILPs is to stimulate uptake of circulating blood sugar and thereby decreasing trehalose levels in the circulation, this study monitored whole-body trehalose levels in fed and starved flies after DTKR knockdown in IPCs. Knockdown of DTKR in IPCs had no effect on trehalose in fed flies, but induced an acute drop in trehalose after 5 h starvation, compared with controls, suggesting an increase in insulin signaling. Analysis of Dilp mutants or knockdown indicated that trehalose levels are regulated by DILP2 (Broughton, 2008; Grönke, 2010); one of the peptides whose transcripts was indeed altered by DTKR knockdown at starvation. In the current experiments lipid levels were not affected by manipulations of DTKR on IPCs in fed or starved flies. Lipid metabolism may be regulated by multiple DILPs, including Dilp6 (Grönke, 2010), or by compensatory AKH signaling, and this may explain the lack of effect after manipulating only IPC activity (Birse, 2011).

Diminishment of DTKR expression on IPCs results in flies that display a shortened lifespan at starvation. This would also indicate increased insulin signaling, as deletion of IPCs or knocking down combinations of DILPs produce the opposite phenotype, and overexpression of Dilp2 in IPCs resulted in decreased resistance to starvation (Enell, 2010). A similar reduction of lifespan at starvation was seen after knock down of the inhibitory GABAB receptor on IPCs (Enell, 2010). It is not clear which of the DILPs regulates the lifespan at dietary restriction or starvation, but DILP2 has been suggested as a candidate (Birse, 2011).

The second known DTK receptor, designated NKD, does not seem to play a role in the regulation of IPCs. NKD can be activated only by one of the DTKs, the N-terminally extended DTK-6, which has not been detected in the Drosophila brain, in contrast to the DTK-1-5 known to activate DTKR (Birse, 2011).

It can be mentioned that an earlier report shows that DTKR is expressed in renal (Malpighian) tubules where it regulates DILP5 signaling (Söderberg, 2011). It is proposed that this regulation is mediated by DTKs released hormonally from endocrine cells of the midgut. DTKs circulating locally act on DTKR expressed in principal cells of the renal tubules, resulting in a local activation of DILP5 signaling. The DTKR-regulated DILP5 signaling in renal tubules does not affect trehalose levels in fed or starved flies, but seems to be part of the defense against oxidative stress (Söderberg, 2011). Thus, this DTK-controlled DILP5 signaling in the tubules is probably independent of the paracrine DTK-mediated IPC regulation in the brain, but further studies of gut-derived DTK action are required to confirm this (Birse, 2011).

In summary these results indicate that in wild-type flies the activated DTKR inhibits insulin signaling in the brain IPCs, and knockdown of the receptor therefore leads to increased insulin signaling. This can be seen in the decreased lifespan and a considerable decrease in trehalose levels during short-term starvation compared with controls, similar to what is expected at increased DILP signaling. The most direct evidence that DTKR is involved in IPC regulation is the effect on Dilp2 and Dilp3 transcript levels seen after receptor knockdown in the IPCs in fed and starved flies. However, it is important for the future to develop a sensitive assay for quantifying hemolymph levels of individual DILPs to monitor how their release is affected by the DTKR signaling to IPCs (Birse, 2011).

The role of classical neurotransmitters in the transfer and processing of olfactory information is well established in many organisms. Neuropeptide action, however, is largely unexplored in any peripheral olfactory system. A subpopulation of local interneurons (LNs) in the Drosophila antennal lobe is peptidergic, expressing Drosophila tachykinins (DTKs). This study shows that olfactory receptor neurons (ORNs) express the DTK receptor (DTKR). Using two-photon microscopy, it was found that DTK applied to the antennal lobe suppresses presynaptic calcium and synaptic transmission in the ORNs. Furthermore, reduction of DTKR expression in ORNs by targeted RNA interference eliminates presynaptic suppression and alters olfactory behaviors. Opposite behavioral phenotypes were detected after reduction and over-expression of DTKR in ORNs. These findings suggest a presynaptic inhibitory feedback to ORNs from peptidergic LNs in the antennal lobe (Ignell, 2009).

Drosophila ORNs express DTKR, which appears to serve in a feedback circuit from local peptidergic interneurons, LNs, of the antennal lobe. These LNs express the peptide products, DTK1-5, of the dtk gene (Winther, 2003). Two-photon imaging and behavioral data, using RNAi and over-expression of the DTK receptor, provide evidence that ORNs are modulated presynaptically by DTKs. This peptidergic presynaptic inhibition of ORNs is detected behaviorally only at high odorant concentrations, and may thus serve to modulate the dynamic range in sensitivity to relevant odors (Ignell, 2009).

Recent studies of peripheral olfactory signal processing in Drosophila have shown that there are afferent and local excitatory cholinergic circuits, combined with presynaptic and postsynaptic GABAergic inhibition of ORNs and interneurons. This study demonstrates a second presynaptic inhibitory pathway mediated by DTK-expressing LNs, providing an additional modulation mechanism in peripheral olfactory processing. Given the importance of presynaptic inhibition future experiments will be necessary to demonstrate a synaptic connection between LNs and ORNs. However, in studies of the cockroach Periplaneta americana both GABAergic and other LNs were found presynaptic to ORNs (Ignell, 2009).

There is ample physiological evidence to suggest that vertebrate and invertebrate ORN axon terminals can be presynaptically modulated by GABA and inhibitory LNs, but this study is unique in that peptidergic presynaptic inhibition of ORNs by local interneurons has been demonstrated. In animals other than Drosophila there is only immunocytochemical data to suggest such circuitry. For example, in the rat, periglomerular cells, interneurons that modulate the first synaptic relay in olfactory processing, have been shown to express 2 neuropeptides: somatostatin and cholecystokinin (Gutierrez-Mecinas, 2005). Moreover, somatostatin receptor immunoreactivity in axons of the rat olfactory nerve has been demonstrated (Schindler, 1999). These morphological studies may suggest that presynaptic peptidergic modulation of ORNs is not exclusive to Drosophila. In addition, there are examples of peptidergic modulation in the olfactory system by efferent neurons. Centrifugal peptidergic modulation has been demonstrated in the olfactory epithelia of a salamander, where neuropeptide Y was shown to enhance responses evoked by a food-related odor in hungry animals (Mousley, 2006). The peptide FMRFamide has been shown to modulate the activity of ORNs in the olfactory epithelium of the mouse and the salamander, but the circuitry is not clear (Ignell, 2009).

Recently, a study of how specific glomeruli mediate olfactory-guided behavior showed that the activation of the DM1 glomerulus is necessary and sufficient to mediate fly attraction to vinegar odor (Semmelhack, 2009). This study tested, physiologically and behaviorally, the response to 2 other food related odors. Both odors elicited activation of DM1 and in flies with down regulated DTKR levels an increased activation of this specific glomerulus was detected. In line with this, it was observed that DTKR knock down flies were more attracted to the 2 odors, suggesting that the behavioral responses to these odors may be linked to DTK mediated inhibition of DM1 (Ignell, 2009).

Neuropeptides often colocalize with classical neurotransmitters in neurons and may act as cotransmitters at synapses. This study found that DTK is expressed in a population partly overlapping with GAD1-expressing LNs, likely to be GABAergic. Therefore, experiments were conducted to determine whether GABA and DTK act synergistically on ORNs. Preliminary data, however, suggest that the 2 compounds may act independently. This is in contrast to findings in the crayfish visual system where GABA hyperpolarizes photoreceptors and a tachykinin-like peptide potentiates this response (Glantz, 2000). In the crayfish, it is not clear which GABA receptor type that mediates the response, although a Cl- conductance appears to be activated, whereas in Drosophila ORNs the presynaptic inhibition is mediated by metabotropic GABAB receptors (Ignell, 2009).

In summary, these findings suggest a presynaptic inhibitory feedback to ORNs from a population of peptidergic LNs in the antennal lobe. These LNs express DTKs, and the ORNs of the antennae express the DTK receptor. Evidence is provided for peptidergic modulation in the antennal lobe before the olfactory signals are relayed via projection neurons to higher brain centers, possibly acting as a mechanism to control olfactory sensitivity (Ignell, 2009).

Neuropeptides related to vertebrate tachykinins have been identified in Drosophila. Two Drosophila G-protein-coupled receptors (GPCRs), designated NKD (CG6515) and DTKR (CG7887), cloned earlier, display sequence similarities to mammalian tachykinin receptors. However, they were not characterized with the endogenous Drosophila tachykinins (DTKs). The present study characterizes one of these receptors, DTKR. It was determined that HEK-293 cells transfected with DTKR displayed dose-dependent increases in both intracellular calcium and cyclic AMP levels in response to the different DTK peptides. DTK peptides also induced internalization of DTKR-green fluorescent protein (GFP) fusion constructs in HEK-293 cells. Specific antireceptor antisera were generated and DTKR was shown to be widely distributed in the adult brain and more scarcely in the larval CNS. The distribution of the receptor in brain neuropils corresponds well with the distribution of its ligands, the DTKs. These findings suggest that DTKR is a DTK receptor in Drosophila and that this ligand-receptor system plays multiple functional roles (Birse, 2006).

Several experimental approaches used in this study have provided data to support the hypothesis that the Drosophila
GPCR, DTKR (CG7887), is involved in signaling by endogenous tachykinin ligands, the DTKs. The most compelling evidence was furnished by the dual assays of Ca22+ and cAMP levels. The assays of intracellular Ca22+ levels indicated that the DTKR receptor, when stably expressed in HEK-293 cells,
displays a distinct activation by all six DTK isoforms. In the assay of cAMP levels, DTKR displayed a distinct activation by DTK-1 to 5 (DTK-6 was not tested). In both cases the responses are clearly dose-dependent (Birse, 2006).

Activation of DTKR was specific to the
six DTK isoforms, because no other peptides that were
tested produced any responses. It appears that DTKR
has a slight preferential affinity for DTK-1, while the
other DTKs display almost an order of magnitude
lower activity levels in the Ca22+
assay. The responses by DTKR to DTKs measured in the cAMP assay
required approximately 10-fold more peptide than
in the Ca22+ assay. Likewise, the stable fly (Stomoxys
calcitrans) tachykinin receptor, STKR, appears to couple
both to changes in Ca22+ levels and in cAMP but with ~10-fold less sensitivity. These results beg the question of whether DTKR normally signals through
cAMP. There are many precedents by which single receptors have been demonstrated to couple to multiple second messengers (Birse, 2006).

When these results are reconsidered with future experiments
in native Drosophila cells, it will be useful to
test the hypothesis that both pathways are regulated by
DTKR activation. In the original identification of
DTKR, insect tachykinins were not known and the
mammalian tachykinin substance P was used at very
high doses to activate the receptor when expressed in
Xenopus oocytes (Li, 1991). The currents monitored
indicated an activation of the PLC pathway, a
signaling pathway commonly recruited by tachykinin
receptors in mammals. It is noteworthy that the mammalian
tachykinins (with FXGLMamide C-terminus) need to
be applied in micromolar concentrations to activate
invertebrate tachykinin GPCRs, whereas invertebrate
TKs (with FXGXRamide) are active even in sub-nanomolar
concentrations on the same receptors (Birse, 2006).

Further support for the role of DTKR as a DTK
receptor derives from ligand-induced GPCR-GFP internalization
experiments. Using HEK-293 cells transfected with a
DTKR-GFP fusion protein internalization
of the receptor could be detected after the application of 1 microM
of DTK-1. Internalization of GPCRs from the plasma
membrane after prolonged activation by specific
ligands is a common phenomenon serving to terminate
peptide activation of the receptor complex. In this process of
desensitization β-arrestin is recruited to the plasma
membrane. An earlier investigation, using a different
approach, demonstrated that a GFP-coupled β-arrestin could be translocated to the plasma membrane of HEK-293 cells transfected with DTKR
and activated by 1 microM of DTK-1 (Johnson,
2003). This technique measures the desensitization of
an activated GPCR regardless of the second messenger
pathway(s) that is activated. DTK-1 specifically activated the DTKR receptor in this
assay, while no response could be seen with any of 12
other unrelated peptides tested (Birse, 2006).

The expression of DTKR was establised in different
portions of adult flies and in the larva by specific
antisera to part of the receptor protein. Immunocytochemistry
indicated that the relative abundance of DTKR and number of immunoreactive cell bodies in the brain are considerably greater than in thoracic
abdominal ganglia both in adults and larvae. The cellular
localization of the DTKR immunoreactivity in
the intestine was hard to resolve, although Western
blotting clearly indicated presence of immunoreactive
material in this tissue. Microarray analysis has shown
that CG7887 transcript is present in low but significant
amounts in Malpighian tubules of flies. However, the current study could not detect reproducible DTKR immunoreactive cells in larval tubules, but
only diffuse labeling in adult tubules (Birse, 2006).

A distinct pattern of distribution of
DTKR immunoreactivity was found in larvae and adults. In the
adult brain the immunolabeling in synaptic neuropil
closely resembles that of arborizing neuronal processes
containing DTKs. Thus there is a good match
between putative release sites of DTKs and the distribution
of their receptor DTKR. The distribution of
DTKs has been studied in Drosophila both by immunocytochemistry
and in situ hybridization in the brain
and intestine (see Siviter, 2000; Nassel, 2002;
Winther, 2003). In the adult brain TK-immunoreactivity
is seen in varicose processes in synaptic
neuropils of the antennal lobe, superior protocerebrum
(including pars intercerebralis), in the central
body, subesophageal ganglion, and in the medulla of
the optic lobe. These brain sites also express tachykinin
receptor protein of DTKR type. The DTKR
receptor distribution would thus suggest that DTKs
and one of their receptors are involved in modulation
of chemosensory and visual processing, control of
hormone release (superior protocerebrum), and modulation
of higher locomotor activities (central body) (Birse, 2006).

In conclusion, this study has shown that one of the
proposed Drosophila tachykinin receptors, DTKR, is
indeed likely to be involved in tachykinin (DTK) signaling
and is expressed widely in the CNS. The
DTKR receptor appears to have a slight preferential
affinity for the DTK-1 isoform compared to DTK-2
to 6 and couples to phospholipase C and to adenylate
cyclase in HEK-293 cells. It would be interesting to
determine the distribution of the other presumed tachykinin
receptor, NKD, to reveal whether the NKD and DTKR receptors display differential distributions in the adult brain and intestine, thus suggesting a functional
and spatial separation of DTK signaling (Birse, 2006).